The overall objective of this proposal is to define the cellular mechanisms culminating in lethal liver cell injury. Because of the importance of tumor necrosis factor-alpha (TNF-alpha) in human liver injury, our program is working on the cellular and subcellular mechanisms causing liver injury by this cytokine. Specifically, our long-term objectives are to understand the mechanisms involved in TNF-alpha-mediated hepatocyte apoptosis. Based on extensive preliminary data, we propose the novel CENTRAL HYPOTHESIS that TNF-alpha signals selective lysosomal permeabilization resulting in cathepsin B release into the cytosol, where it initiates mitochondrial dysfunction resulting in apoptosis and liver injury. We will now employ current and complementary, molecular, biochemical and cell biological approaches to ascertain how TNF-alpha triggers this unexplored pathway of apoptosis. Our proposal has three SPECIFIC AIMS. FIRST, we will directly test the hypothesis that TNF-alpha signal lysosomal permeabilization: a) by activating neutral sphingomyelinases through the adaptor protein FAN (factor associated with neutral sphingomyelinase) with subsequent sphingosine generation; and b) by a process dependent upon intra-lysosomal cathepsin B (i.e., cathepsin B-dependent cathepsin B release). SECOND, we will test the hypothesis that cytosolic cathepsin B mediates apoptosis: a) by activating the mitochondrial pathway of apoptosis; and b) by cleaving the proapoptotic member of the Bcl-2 family Bid, resulting in its activation and translocation to mitochondria where it induces cytochrome c release. FINALLY, we will test the hypothesis that cathepsin B mediates liver injury such that: a) cathepsin B knockout mice are resistant to liver injury following endogenous stimulation of TNF-alpha in the bile duct-ligated mouse; and b) cathepsin B knockout mice have reduced parameters of liver fibrosis following bile duct ligation. The proposal is innovative, technically and conceptually, as it tests new concepts for TNF-alpha mediated liver injury and apoptosis in general using sophisticated technologies. The significance of the information generated is that it will provide a framework for the potential development of novel therapeutic strategies effective in attenuating human liver injury.